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Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane
Journal article   Open access   Peer reviewed

Positive charges in a putative amphiphilic helix in the carboxyl-terminal region of the third intracellular loop of the luteinizing hormone/chorionic gonadotropin receptor are not required for hormone-stimulated cAMP production but are necessary for expression of the receptor at the plasma membrane

HAIYUN WANG, Julie Jaquette, Kimberly Collison and Deborah L Segaloff
Molecular endocrinology (Baltimore, Md.), Vol.7(11), pp.1437-1444
1993
DOI: 10.1210/mend.7.11.8114758
PMID: 8114758
url
https://doi.org/10.1210/mend.7.11.8114758View
Published (Version of record) Open Access

Abstract

The LH/CG receptor (LHR) is a member of the family of G protein-coupled receptors and activates Gs when stimulated by LH or CG. Studies from other G protein-coupled receptors have implicated the carboxyl-terminal region of the third intracellular loop as being involved in the activation of G proteins. It has been suggested that the potential ability of this region to form an amphiphilic helix, with positively charged residues aligned to one face, may be important for this biological activity. To test whether the positively charged lysine residues, and thus an amphiphilic helix, in the carboxyl terminal region of the rat LHR (rLHR) are indeed important in the activation of Gs by the rLHR, a mutant rLHR was constructed in which lysines 541, 544, and 557 were simultaneously substituted with alanines. Clonal 293 cells expressing comparable numbers of cell-surface recombinant wild type rLHR or rLHR(K541, 544, 547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated cAMP production could readily be interpreted as an alteration in the mutant receptor's ability to activate Gs. It was found, however, that basal cAMP production, the concentration of hCG required to elicit half-maximal cAMP production, and the maximal levels of cAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541, 544, 547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated CAMP production could readily be interpreted as an alteration in the mutant receptor’s ability to activate Gs. It was found, however, that basal CAMP production, the concentration of hCG required to elicit half-maximal CAMP production, and the maximal levels of CAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541, 544, 547A). Although these data do not necessarily refute the role of the carboxyl-terminal portion of the third intracellular loop of the rLHR in activating Gs, they do indicate that neither an amphiphilic helix nor the positively charged residues in this region per se are required for this activity. We also found that substitution of these lysine residues with alanines caused an impairment in the trafficking of the receptor to the plasma membrane.
 Gene Expression Molecular Genetics Fundamental and applied biological sciences. Psychology Biological and medical sciences Molecular and cellular biology

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